**Stool testing**


In fact, blood testing should include complete blood cell count, liver enzyme, creatinine, and C-reactive protein to check overall clinical condition, serology for Hepatitis virus, and Human immunodeficiency virus (HIV). Furthermore, blood tests can be considered in case of anomalies of the first round of laboratory tests, endemic spread of some pathogens, emergence of new pathogens or selected cases of recipients (e.g., immunosuppressed). In particular, there is debate about the usefulness of serology for EBV and CMV, as the high prevalence of prior exposure among adult individuals weakens the diagnostic power of this approach, limiting the clinical utility to IgM CMV in donors dedicated to immunosuppressed recipients. Of course, it is not appropriate to exclude subjects with prior exposure to EBV or CMV from the donation because of the unlikely risk of transmission, unless clinical or laboratory suspicion of reactivation. Finally, the candidates could be considered for testing the serology for nematodes, based on social and geographical features and tests availability [13].

Stool testing should include common enteric pathogens, *Clostridium di*ffi*cile*, fecal parasites, and *Helicobacter pylori* antigen (this last exam only for upper route of FMT delivery). Enteric pathogens, which must also be investigated in asymptomatic subjects, should be detected with conventional methods (culture, microscopy, or antigen test) and/or with molecular diagnosis (PCR-based panels) that have shown a high specificity and sensitivity compared to conventional methods in rapid detection of pathogens [60]. Furthermore, it is mandatory to test all fecal samples for antibiotic-resistant bacteria (including meticillin-resistant *Staphylococcus aureus* (MRSA), vancomycin-resistant Enterococci (VRE), extended-spectrum β-lactamase-producing Enterobacteriaceae, and carbapenem-resistant Enterobacteriaceae/carbapenemase-producing Enterobacteriaceae), considering the burden of the gastrointestinal carriage in asymptomatic subjects [61,62] and the reporting of some serious adverse events associated to sepsis after FMT [13]. Nowadays, due to the emerging Covid-19 pandemic, a panel of international experts has suggested to include in the tests for Sars-CoV-2 a thorough nasopharyngeal swab and/or RNA detection in stool [63].

Finally, if all blood and fecal tests are negative, the candidate is accepted to become a stool donor. Especially in the fecal bank program, the donor should be available to donate on many occasions over time. For this reason, it is advisable to repeat the screening tests every 8–12 weeks and administer a short questionnaire on the same day of the donation to check for any recent-onset harmful events.

In this paragraph we have reported the general rules to select and to screen potential donor for FMT, mainly to treat CDI that is cured by the restorative effect of fecal transfer on gu<sup>t</sup> microbiota. However, for other clinical indications, which find their rationale in the modification of metabolic and

inflammatory pathways mediated by gu<sup>t</sup> microbiota, it would be appropriate to identify a specific donor for each case. This issue will be discussed later.

#### **4. Selection of the Optimal Donor for FMT to Treat Specific Disorders**

The correct recruitment of healthy donors is essential for a standardized and safe FMT procedure [11,13]. FMT is considered a safe procedure; however, mild adverse e ffects attributable to FMT are reported in about one third of the recipients, such as self-limiting abdominal discomfort or changes of bowel habits, and unfortunately, about 2–6% of patients experienced serious adverse events, such as infection, relapse of pre-existing disease, or death [64]. Moreover, the di fficulty of selecting the appropriate candidates is increasing due to emerging concerns, as the possibility of transmission of putative procarcinogenic bacteria [65] or the potential risk of serious life threatening infections with multi-drug resistant organisms after FMT [66]. Moreover, recent evidences showed that the e fficacy of FMT in recurrent CDI treatment, in clinical trials and in other healthcare settings seems to be linked to di fferent variables, such as the delivery methods of fecal infusate, the bowel preparation, the number of infusion, the disease severity, and in particular to the microbial diversity and composition of the transplanted stools [32,44,67]. Since the idea that the success rate of FMT could be related to the gu<sup>t</sup> microbiota or other features of the donor, the term "super-donors" has been introduced to indicate the ideal individuals whose stools could ensure a better outcome for recipients compared to others fecal donations [68]. Therefore, assuming that dysbiosis-related disorders have been associated to di fferent imbalanced microbial signatures [15], in order to restore the eubiosis, it is reasonable to assume that reaching the correct donor-recipient match with targeted FMT based on specific microbial disturbances might be the key to improve FMT response. Accumulating evidence strengthens this hypothesis, leading to discard the concept of "one stool fits all" and to search an optimal donor [68], as in other organ transplantation procedures [69].

#### *4.1. Clostridium Di*ffi*cile Infection*

The research of the ideal donor in recurrent CDI is obviously a widely debated topic of study. For example, one study identified the optimal donor among nine healthy vegetarian or vegan candidates, selecting the candidate who had a balanced Bacteroidetes/Firmicutes ratio, the highest alpha diversity among screened individuals, and high butyrate concentration. After 10 weeks from a single or multiple FMT, none of the 10 patients experienced CDI recurrence [70]. Of interest, the gu<sup>t</sup> virome may also play a role in CDI treatment [71]. Indeed, enteric virome alterations marked by an increase in the abundance of *Caudovirales*, together with a decreased *Caudovirales* diversity, richness, and evenness, have been reported in patients with CDI. Moreover, CD eradication was associated with the colonization of a higher abundance of donor-derived *Caudovirales* contigs detected during follow up. These findings could possibly explain why bacterial fecal filtrate infusion resulted in e ffective treatment of CDI [72], and shifted the attention on the importance of the bacteriophages and on the potential role of selecting donors on the basis of their gu<sup>t</sup> virome. Finally, some authors reported that selecting specific enteric bacterial strains with bacterial cultures from healthy donors to prepare a stool substitute blend might be a winning strategy to cure recurrent and antibiotic-resistant *C. di*ffi*cile* colitis [73,74]. However, it is likely that the relevant impact on FMT success in CDI depends on the transfer of a complete fecal microbiome rather than specific bacterial strains; moreover, the promising results reported by the study that transfer the fecal filtrate alone sugges<sup>t</sup> a predominant role for bacteriophages rather than for the specific relative abundance pattern of the gu<sup>t</sup> microbiota of donor, shifting the central role from bacteria to viruses in the therapeutic challenge of FMT in CDI; however, these data are still preliminary and need to be confirmed by further studies.

#### *4.2. Inflammatory Bowel Disease*

Many studies analyzed the microbial profile of donors and tried to relate it with clinical and laboratory outcomes in patients with IBD. Clinical outcomes and immunological changes after FMT in patients with IBD were significantly related to the variations of several specific strains in recipients of fecal microbiota [75]. For instance, intensive FMT in UC patients were associated with negative outcomes in case of abundance of *Fusobacterium spp* and *Sutterella spp* in recipients' fecal microbiota after the FMT [76]. Furthermore, a study that involved refractory UC patients reported that pre-treatment with antibiotic plus repeated FMTs using fecal material from donor with a high bacterial richness and high relative abundance of *Akkermansia muciniphila*, unclassified Ruminococcaceae, and *Ruminococcus spp*. was more likely to induce remission compared to antibiotics alone [67]. As also described in other studies [41,77,78], it is plausible that choosing donors based on their taxonomic composition, in particular low or high abundance of specific strains, might reflect the possibility for future trials in IBD. For this purpose, methods aimed at preventing an inflammatory response of the recipient's intestinal immune system by selecting compatible donors on their microbial profiles are under study [79]. Furthermore, the gu<sup>t</sup> virome could represent a potential marker for FMT response in UC patients. In particular, results from a small case series reported that FMT responders already presented, before undergoing to FMT, a significantly lower eukaryotic viral richness than non-responders. Moreover, the richness of donor virome was not associated with the FMT outcome, as instead proposed for bacteria [80].

#### *4.3. Other Emerging Indications*

Several preclinical and clinical studies supported the rationale for donor selection based on gu<sup>t</sup> microbial profile in other disorders associated to gu<sup>t</sup> dysbiosis. Indeed, in the field of anti-cancer treatment, it has been reported that microbiota can influence chemotherapy response [81]. Preclinical studies found a clinical improvement in mouse models of melanoma on anti-PD-1 therapy that received FMT from donors with a melanoma "responder-like" microbial signature (with high alpha diversity and abundance of Ruminococcaceae, *Faecalibacterium*, *Bifidobacterium longum*, *Collinsella aerofaciens*, and *Enterococcus faecium*) when compared to mice that received "non responder-like" microbiome (characterized by low microbial diversity and high relative abundance of Bacteroidales) [82,83]. Nevertheless, trials on humans, testing the e ffect of FMT in increasing the response to cancer therapies, are still in progress [84].

Recently, a randomized placebo-controlled trial of FMT in IBS reported that the abundance of *Streptococcus*, *Dorea*, *Lactobacillus*, and *Ruminococcaceae* spp in the donor microbiota was associated with e fficacy in relieving IBS symptoms [44]. Interestingly, a small open-label clinical trial evaluated the impact of prolonged FMT with antibiotic pre-treatment in children with autism; authors reported a decrease of gastrointestinal symptoms and an improvement of behavior, together with specific genera increase in recipients (*Bifidobacterium*, *Prevotella*, and *Desulfovibrio*). Conversely, *Prevotella*, and *Desulfovibrio* were more represented in recipients after FMT than in the donor samples, suggesting that unknown factors changed the intestinal ecosystem, making it more hospitable to these strains [85].

Within the context of metabolic diseases, the e ffect of allogenic FMT post-Roux-en-Y gastric bypass donors was compared with metabolic syndrome donors on glucose metabolism and other parameters in treatment-naïve patients with metabolic syndrome. The authors assessed a decrease of insulin sensitivity in recipients who received FMT from donors with metabolic syndrome compared with using post-surgical donors. Moreover, they identified several microbial OTUs possibly predictive of metabolic response, suggesting a microbiota-related transmissible mechanism of insulin resistance [86]. Similarly, another study reported a significant increase in insulin sensitivity, together with altered microbiota composition, in patients with metabolic syndrome who received allogenic FMT from lean donors compared to those who underwent autologous FMT [46].

To date, these results appear promising but partially controversial; thus, findings need to be confirmed with stronger evidence and by standardized clinical trial. Further research is needed to identify the favorable microbial signature of donor or other ideal features in disease-specific settings.

#### **5. Conclusions and Future Perspectives**

In this review, the stool donor screening process has been described, and recent evidence has been reported that try to identify the optimal donor for each clinical condition (Figure S1).

To date, the clinical characteristics of the donor are well defined; in particular, they are recommended to be a healthy volunteer with a balanced lifestyle, without chronic diseases or family history of metabolic diseases or cancer, and defined laboratory exams must certify the current absence of disease. However, identification of the ideal donor through the microbiological typing of the stool is currently not suitable. First of all, understanding the role of the intestinal microbiota in each chronic disease is an indispensable condition before hypothesizing a personalized approach through FMT. In fact, while the restorative mechanism of FMT in recurrent CDI is now understood, many aspects still need to be understood regarding the treatment of other chronic conditions. Interesting evidence has been reported regarding dysbiosis in IBD or in other chronic conditions, but the contrasting results reported in clinical trials of FMT could be justified by the choice of unsuitable donors. The identification of the microbiological characteristics of the ideal donor for each disease appears to be an achievable goal but still far from being accomplished due to the lack of clinical studies. The current evidence is still limited and insu fficient for explaining and resolving the complexity of the interaction between the intestinal barrier and its role in gut-related chronic diseases. However, further studies need to be designed to confirm the encouraging results that have been reported in recent years. In particular, it will be necessary to type the fecal microbiota of the donor and the recipient, and to understand how environmental factors, such as diet, or individual features may benefit (or not) the clinical response to FMT. Understanding the microbial characteristics of the optimal donor, in particular if they are modifiable through lifestyle changes or pharmacological measures, could increase the therapeutic potential of FMT.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/6/1757/s1, Figure S1: Optimal Stool Donor to Treat Specific Disorders.

**Author Contributions:** Conceptualization, S.B. and A.G.; data curation, S.B., C.R.S. and S.P.; writing—original draft preparation, S.B. and C.R.S.; writing—review and editing, S.B., C.R.S., S.P., E.B., G.I., G.C. and A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
